Attila Jenei

Colocalization and nonrandom distribution of Kv1.3 potassium channels and CD3 molecules in the plasma membrane of human T lymphocytes

Distribution and lateral organization of Kv1.3 potassium channels and CD3 molecules were studied by using electron microscopy, confocal laser scanning microscopy, and fluorescence resonance energy transfer. Immunogold labeling and electron microscopy showed that the distribution of FLAG epitope-tagged Kv1.3 channels (Kv1.3/FLAG) significantly differs from the stochastic Poisson distribution in the plasma membrane of human T lymphoma cells. Confocal laser scanning microscopy images showed that Kv1.3/FLAG channels and CD3 molecules accumulated in largely overlapping membrane areas. The numerical analysis of crosscorrelation of the spatial intensity distributions yielded a high correlation coefficient (C = 0.64). A different hierarchical level of molecular proximity between Kv1.3/FLAG and CD3 proteins was reported by a high fluorescence resonance energy transfer efficiency (E = 51%). These findings implicate that reciprocal regulation of ion-channel activity, membrane potential, and the function of receptor complexes may contribute to the proper functioning of the immunological synapse.

Complexity of signal transduction mediated by ErbB2: clues to the potential of receptor-targeted cancer therapy

The erbB2 oncogene belongs to the type I transmembrane tyrosine kinase family of receptors. Its medical importance stems from its widespread overexpression in breast cancer. This review will focus on the signal transduction through this protein, and explains how the overexpression of erbB2 may result in poor prognosis of breast cancer, and finally it will summerize our current understanding about the therapeutic potential of receptor-targeted therapy in breast cancer. ErbB2 does not have any known ligand which is able to bind to it with high affinity. However the kinase activity of erbB2 can be activated without any ligand, if it is overexpressed, and by heteroassociation with other members of the erbB family (erbB1 or epidermal growth factor receptor, erbB3 and erbB4). This interaction substantially increases the efficiency and diversity of signal transduction through these receptor complexes. In addition, erbB2 forms large scale receptor clusters containing hundreds of proteins. These receptor islands may take part in recruiting cytosolic factors which relay the signal towards the nucleus or the cytoplasm. Overexpression of erbB2 was linked to higher transforming activity, increased metastatic potential, angiogenesis and drug resistence of breast tumor in laboratory experiments. As a corollary of these properties, erbB2 amplification is generally thought to be associated with a poor prognosis in breast cancer patients. These early findings lead to the development of antibodies that down-regulate erbB2. Such a therapeutic approach has already been found effective in experimental tumor models and in clinical trials as well. Further understanding of the importance of erbB2 and growth factor receptors in the transformation of normal cells to malignant ones may once give us a chance to cure erbB2 overexpressing breast cancer.

Understanding FRET as a Research Tool for Cellular Studies

Communication of molecular species through dynamic association and/or dissociation at various cellular sites governs biological functions. Understanding these physiological processes require delineation of molecular events occurring at the level of individual complexes in a living cell. Among the few non-invasive approaches with nanometer resolution are methods based on Förster Resonance Energy Transfer (FRET). FRET is effective at a distance of 1–10 nm which is equivalent to the size of macromolecules, thus providing an unprecedented level of detail on molecular interactions. The emergence of fluorescent proteins and SNAP- and CLIP- tag proteins provided FRET with the capability to monitor changes in a molecular complex in real-time making it possible to establish the functional significance of the studied molecules in a native environment. Now, FRET is widely used in biological sciences, including the field of proteomics, signal transduction, diagnostics and drug development to address questions almost unimaginable with biochemical methods and conventional microscopies. However, the underlying physics of FRET often scares biologists. Therefore, in this review, our goal is to introduce FRET to non-physicists in a lucid manner. We will also discuss our contributions to various FRET methodologies based on microscopy and flow cytometry, while describing its application for determining the molecular heterogeneity of the plasma membrane in various cell types.

Modification of membrane cholesterol level affects expression and clustering of class I HLA molecules at the surface of JY human lymphoblasts

Recently we have found that class I HLA molecules, key elements of the antigen presentation system for CD8 + effector cells, show a clustered lateral distribution (homoassociation) at the surface of activated human T- and B-lymphocytes as well as virus-transformed T- and B-lymphoblasts, in contrast to a disperse distribution on resting human PBLs (Matk6 et al. (1994) J. Immunol. 152, 3353; Bene et al. (1994) Eur. J. Immunol. 24, 2115). Expression of beta2m-free HLA heavy chains and exogenous beta2m have been shown as potential regulation factors of HLA-I clustering, which in turn may affect cytotoxic activity of CD8+ effector cells. Here we report a study on the effect of plasma membrane-modification (by exogenous cholesterol and phosphatidylcholine) on the expression of free HLA heavy chains and beta2m-bound HLA-I molecules on JY human B-lymphoblasts. The modulating effect of these two treatments on the lipid fluidity of cells was demonstrated by fluorescence anisotropy of DPH lipid probe. The lateral clustering (association) of HLA-I molecules was detected by flow cytometric fluorescence resonance energy transfer (FCET) and digital imaging microscopic photobleaching energy transfer (pbFRET) methods, using flourescein-isothiocyanate (FITC) (donor)- and tetramethyl-rhodamine-isothiocyanate (TRITC) (acceptor)-labeled W6/32 or KE2 antibodies directed against intact HLA-I molecules. Cholesterol enrichment of the plasma membrane increased membrane fluidity and reduced the expression of heavy- and light-chain determinants of HLA-I molecules and free heavy chains (FHCs). This was accompanied with a higher degree of HLA-I clustering as shown by the enhanced intermolecular energy transfer efficiency. In contrast, cholesterol depletion resulted in membrane fluidization and increased expression of HLA-I epitopes. Our results suggest that both cholesterol level and lipid structure/fluidity of the plasma membrane in lymphoblastoid cells may also potentially regulate lateral organization and consequently the presentation efficiency of HLA-I molecules.

PICOSECOND MULTIPHOTON SCANNING NEAR-FIELD OPTICAL MICROSCOPY

We have implemented simultaneous picosecond pulsed two- and three-photon excitation of near-UV and visible absorbing fluorophores in a scanning near-field optical microscope (SNOM). The 1064-nm emission from a pulsed Nd:YVO4 laser was used to excite the visible mitochondrial specific dye MitoTracker Orange CM-H2TMRos or a Cy3-labeled antibody by two-photon excitation, and the UV absorbing DNA dyes DAPI and the bisbenzimidazole BBI-342 by three-photon excitation, in a shared aperture SNOM using uncoated fiber tips. Both organelles in human breast adenocarcinoma cells (MCF 7) and specific protein bands on polytene chromosomes of Drosophila melanogaster doubly labeled with a UV and visible dye were readily imaged without photodamage to the specimens. The fluorescence intensities showed the expected nonlinear dependence on the excitation power over the range of 5-40 mW. An analysis of the dependence of fluorescence intensity on the tip-sample displacement normal to the sample surface revealed a higher-order function for the two-photon excitation compared to the one-photon mode. In addition, the sample photobleaching patterns corresponding to one- and two-photon modes revealed a greater lateral confinement of the excitation in the two-photon case. Thus, as in optical microscopy, two-photon excitation in SNOM is confined to a smaller volume.

Strength in Numbers: Effects of Acceptor Abundance on FRET Efficiency

Fluorescence resonance energy transfer (FRET) is a strongly distance-dependent process between a donor and an acceptor molecule, which can be used for sensitive distance measurements and characterization of molecular interactions at the nanometer level. The original mathematical description of this process, however, is only valid for the interaction of one donor with one acceptor. This criterion is not always met, especially in biological systems, where multiple structures can interact simultaneously, often making distance estimations based on transfer efficiency values error-prone. Herein we investigate how the interaction of multiple acceptors and donors influences the transfer efficiency value in an intramolecular cellular FRET system by manipulating the fluorophore/protein ratio of the fluorophore-conjugated antibodies. We show that the labeling ratio of the acceptor has the largest influence on measured transfer efficiency and decreasing or increasing the acceptor labeling ratio can be utilized to manipulate the FRET response of the acceptor-donor pair and therefore is a tool for optimizing sensitivity of FRET measurements.

Fluorescence resonance energy transfer detected by scanning near‐field optical microscopy

Fluorescence resonance energy transfer (FRET) between excited fluorescent donor and acceptor molecules occurs via the Förster mechanism over a range of 1–10 nm. Because of the strong (sixth power) distance dependence of the signal, FRET has been used to assess the proximity of molecules in biological systems. We used a scanning near‐field optical microscope (SNOM) operated in the shared‐aperture mode using uncoated glass fibre tips to detect FRET between dye molecules embedded in polyvinyl alcohol films and bound to cell surfaces. FRET was detected by selective photobleaching of donor and acceptor fluorophores. We also present preliminary results on pixel‐by‐pixel energy transfer efficiency measurements using SNOM.

Ion-channel activities regulate transmembrane signaling in thymocyte apoptosis and T-cell activation.

Several examples have shown that plasma membrane ion channels (e.g., Ca2+ and K+ channels) make an important contribution to lymphocyte activation or thymocyte apoptosis. Here we report on the importance of these ion channels in the sensitivity or resistance of lymphoid cells to extracellular ATP-induced apoptosis. Thymocytes of Balb/c mice responded to extracellular ATP (ATPex) sensitively, with an immediate increase in the intracellular calcium level and later with an increased membrane permeability to low MW markers. Mature (medullary) thymocytes showed a higher sensitivity than did cortical thymocytes. Three human lymphoma cell lines, including SUPT13, a cell line reported to be sensitive to TcR/CD3 activation-induced apoptosis, showed a high resistance to ATPex action. These observations suggest that maturation/differentiation state-dependent activity or disappearance of early ATP-receptor operated signaling systems (including ion channels) are critical for the cells in developing towards apoptosis. Using the patch-clamp technique we demonstrated that bretylium tosylate (a particular K(+)-channel blocker) known as inhibitor of T-lymphocyte proliferation also influences the single-channel properties of voltage-gated K+ channels through depressing whole-cell K+ currents. This finding is yet another example underlying the importance of K+ channel activity in T-lymphocyte proliferation.

Angiotensin II increases the permeability and PV-1 expression of endothelial cells

Angiotensin II (ANG II), the major effector molecule of the renin-angiotensin system (RAS), is a powerful vasoactive mediator associated with hypertension and renal failure. In this study the permeability changes and its morphological attributes in endothelial cells of human umbilical vein (HUVECs) were studied considering the potential regulatory role of ANG II. The effects of ANG II were compared with those of vascular endothelial growth factor (VEGF). Permeability was determined by 40 kDa FITC-Dextran and electrical impedance measurements. Plasmalemmal vesicle-1 (PV-1) mRNA levels were measured by PCR. Endothelial cell surface was studied by atomic force microscopy (AFM), and caveolae were visualized by transmission electron microscopy (TEM) in HUVEC monolayers. ANG II (10(-7) M), similarly to VEGF (100 ng/ml), increased the endothelial permeability parallel with an increase in the number of cell surface openings and caveolae. AT1 and VEGF-R2 receptor blockers (candesartan and ZM-323881, respectively) blunted these effects. ANG II and VEGF increased the expression of PV-1, which could be blocked by candesartan or ZM-323881 pretreatments and by the p38 mitogem-activated protein (MAP) kinase inhibitor SB-203580. Additionally, SB-203580 blocked the increase in endothelial permeability and the number of surface openings and caveolae. In conclusion, we have demonstrated that ANG II plays a role in regulation of permeability and formation of cell surface openings through AT1 receptor and PV-1 protein synthesis in a p38 MAP kinase-dependent manner in endothelial cells. The surface openings that increase in parallel with permeability may represent transcellular channels, caveolae, or both. These morphological and permeability changes may be involved in (patho-) physiological effects of ANG II.

Mapping of cell surface protein-patterns by combined fluorescence anisotropy and energy transfer measurements

LIMBURGS UNIV CTR,PHYSIOL RES GRP,B-3590 DIEPENBEEK,BELGIUM.MATKO, J, DEBRECEN UNIV MED,SCH MED,DEPT BIOPHYS,POB 3,H-4012 DEBRECEN,HUNGARY.